We have several research programs, each investigating and exploiting the important and exquisite reactivities of redox-active sites and metal centres (Fe, Ni, Cu, Mo) in enzymes. We are developing new directions and applying a range of powerful physical techniques, particularly dynamic electrochemical methods, EPR spectroscopy and light-driven enzyme catalysis on nanoparticles. We have several goals.

One goal is to establish fundamental chemical directions for future renewable energy technologies – an area of intense innovation that is now at the forefront of international science. Capturing the Sun’s energy as a storable chemical (fuel) is a well-known characteristic of life and we are building on the chemistry that is involved, exploring new avenues and inventing technologies based on enzymes. Enzymes are the most efficient electrocatalysts and a wonderful inspiration for us in terms of what is possible! Enzymes can convert interconvert H2, O2 and water with the highest rates and minimum of energy waste, they can activate CO2 – producing CO for fuel and for organic chemistry, with just a tiny overpotential. The need to optimise efficiency as well as rates when considering how an organism uses the energy available to it has driven the evolution of ‘near perfect’ enzymes. We are using a suite of novel electrochemical techniques, protein film electrochemistry, developed in Oxford, to study how enzymes such as hydrogenases (Fe,Ni), 'blue Cu' oxidases (Cu) and carbon-monoxide dehydrogenases (Fe,Ni) achieve rates that are orders of magnitude higher than conventional catalysts. We are using these enzymes in novel fuel cells that can even produce electricity from low H2 levels in air; we are attaching pairs of enzymes to conducting particles to produce novel catalysts; we are attaching enzymes to semi-conductor nanoparticles to harness sunlight and convert water into H2 and CO2 into CO. All these investigations reveal how human lifestyles could be changed for the better by being able to design catalysts as good as enzymes – a point we refer to as ‘bio-inspired catalysis’.

We are investigating how hydrogenases, enzymes whose active sites are essentially ‘organometallic’ compounds deeply buried and protected within the protein, should be able to function in the presence of O2. This is important for developing strains of microorganisms that could be farmed to produce H2 from sunlight using photosynthesis.

We are studying how the hydrogenases produced by human pathogens, including common bacteria such as E. coli but also Salmonella, are important in maintaining these organisms’ virulence. Hydrogen plays a major role in the energy-producing reactions of microorganisms.

The biohydrogen production project: In this project we are elucidating the mechanistic principles of H2 production by hydrogenases. Many groups worldwide are now interested in ‘Bio Hydrogen’. This process occurs mainly in strictly anaerobic microbes. [FeFe]-hydrogenases, which are produced by green algae, are very O2-sensitive, on the other hand, [NiFe]-hydrogenases produced by cyanobacteria, have a mechanism for fending off and repairing damage by O2. We are studying the basis for O2 sensitivities to establish how organisms could be engineered to function in photosynthetic H2 production.

The enzyme fuel cell project: Hydrogen is the transportable fuel of the future, offering clean and renewable energy. In this research, our studies on [NiFe]-hydrogenases and blue Cu oxidases are being applied with the aim of producing a fuel cell that is 100% derived from biological catalysts, and does not include any expensive chemicals or rare elements as redox mediators. We are able to build miniature low-power fuel cells that if developed for stability, could be used to power electronic devices. For these gadgets to work, it is necessary to use ‘O2-tolerant’ hydrogenases, and are working with a [NiFe]-hydrogenase (Hyd-1) from E.coli that we are producing and engineering in the laboratory. We are working closely with experts in microbiology - Prof. Bärbel Friedrich, of the Humboldt University in Berlin, and Prof. Frank Sargent at the University of Dundee.

Carbon dioxide activation by enzymes using electrical energy and sunlight: We are studying an intriguing class of metalloenzymes known as carbon monoxide dehydrogenases (CODHs) that can interconvert CO and CO2 with great efficiency, providing an important benchmark and challenge for the development of conventional catalysts. CODHs from anaerobic bacteria contain a very unusual [Ni4Fe‑5S] cluster. In contrast to small molecule catalysts, CODH converts CO2 rapidly (and reversibly) into CO at a potential of about -0.5 V at neutral pH. This is a reaction of great technological importance. Otherwise (at synthetic catalysts) the process is so inefficient and slow that it has made little impact on industry. We are working closely with Prof. Steve Ragsdale at the University of Michigan, to develop ways of using this enzyme. Recent breakthroughs include a particulate catalyst for the water-gas-shift (WGS) reaction and a nanoparticle technology that uses sunlight to convert CO2 into CO.

Enzyme catalysis at conducting particles and semi-conductor nanoparticles: In this project we are taking enzyme electrochemistry to the particulate and nanotechnology world, co-attaching enzymes with partner enzymes to make new kinds of catalysts and with photosensitising metal complexes on semi-conducting nanoparticles to activate H2O and CO2 to produce H2 and CO using sunlight as energy source.

Novel biomimetic electrode surfaces: We are devising ways to attach enzyme molecules to carbon and other electrode/semi-conductor surfaces using strong covalent and non-covalent linkages and in such a way as to allow fast electron transfer to and from their active sites. These modifications are then applied in the fuel cell and particles projects.

Oxidising centres in proteins: ‘Blue’ Cu oxidases and Fe(IV)=O: So-called ‘blue’ Cu oxidases from fungi (called laccases) carry out four-electron reduction of O2 to water at potentials quite close to the reversible value (> 0.8 V at pH 5). These centres are not only of fundamental interest, but these enzymes are also electrocatalysts for enzyme-based fuel cells. Another area of interest is the ferryl group Fe(IV)=O, which is an important yet elusive intermediate in the catalytic cycles of many oxidative enzymes, such as cytochrome c peroxidase. We are using protein film electrochemistry to study the properties of Fe(IV) species formed as catalytic intermediates.

Electron Paramagnetic Resonance (EPR) Spectroscopy investigations of metalloenzymes and their binding to surfaces. As part of the Oxford Centre for Electron Spin Resonance (CAESR) we are using EPR techniques, including advanced methods such as DEER, in collaboration with Dr Jeff Harmer, to study reactions of the active sites of metalloenzymes and investigate the interaction of these enzymes at the surfaces of nanoparticles. A recent project of considerable importance is an investigation of the FeS clusters in mitochondrial Complex I, carried out in collaboration with Dr Judy Hirst at the The Medical Research Council Mitochondrial Biology Unit in Cambridge.

The mechanisms of redox catalysis by complex multi-centred electron-transport enzymes: We are using protein film electrochemistry along with other methods to study complex multi-centred metalloenzymes. Many of these have medical importance and technological applications, as well as being of fundamental interest. Examples include respiratory enzymes like succinate dehydrogenase (found in mitochondria), different fumarate reductases, nitrate reductase, and DMSO reductase, as well as peroxidases and hydrogenases. Recently we discovered that aldehydes inhibit H2 production by [FeFe]-hydrogenases, and this is providing important new mechanistic insight.

Metal-sulfide clusters in proteins: Metal-sulfide clusters occur in virtually all forms of life, being found in numerous enzymes and other proteins. Functions include electron transfer, redox catalysis, Lewis acid catalysis, and sensors of Fe levels and oxidative stress. They are now established as agents of metabolic regulation and gene expression. Our goal is to understand the chemistry of clusters: how they are assembled and fragmented, how they bind acids (metal ions, protons) and bases (ligands, substrates), and the factors that determine their complex redox properties. In conjunction with Prof. Juan Fontecilla-Camps (Grenoble), and Prof. Bärbel Friedrich (Berlin), we have determined the structure and properties of a new type of FeS cluster, a [4Fe-3S] cluster coordinated by six cysteines, that can transfer TWO electrons to rescue the active site of [NiFe]-hydrogenases from attack by O2. This property forms a key part of the basis for O2 tolerance of hydrogenases in certain organisms.

Mechanism and energetics of long-range electron-transfer and proton-transfer reactions of metalloproteins and how these are ‘gated’: Fourier transform voltammetry is an electrochemical technique invented in the group of Prof. Alan Bond, at Monash University, Australia. We are collaborating with Alan Bond to exploit this technique to uncover the complex reactions of redox centres in proteins. Electron transfer (ET) and proton transfer (PT) are essential processes in biological systems. We are studying how ET is controlled by the properties of the redox centres, how electrons are relayed by chains of redox-active groups, including amino-acids, and how the electromotive energy (nEF) is used to drive other ‘coupled’ processes. These include formation of covalent bonds (catalysis), changes in protein conformation, and PT which is the basis of energy transduction in membrane-bound respiratory enzymes called proton pumps. Proton tunneling occurs over much shorter distances than electron tunneling, so that in a protein, PT requires closely spaced water molecules or amino acids as mediators and couriers. ET reactions which are controlled by the rates of coupled processes such as PT are said to be ‘gated’. Fast-scan and Fourier transform voltammetry enables complex ET and coupled PT reactions to be studied and deconvoluted in the sub-millisecond time domain. We can make detailed comparisons between structurally characterised metalloproteins and genetically engineered mutant forms in which specific residues have been replaced.